One-Pot Radiosynthesis of [18F]Anle138b—5-(3-Bromophenyl)-3-(6-[18F]fluorobenzo[d][1,3]dioxol-5-yl)-1H-pyrazole—A Potential PET Radiotracer Targeting α-Synuclein Aggregates

Availability of PET imaging radiotracers targeting α-synuclein aggregates is important for early diagnosis of Parkinson’s disease and related α-synucleinopathies, as well as for the development of new therapeutics. Derived from a pyrazole backbone, 11C-labelled derivatives of anle138b (3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole)—an inhibitor of α-synuclein and prion protein oligomerization—are currently in active development as the candidates for PET imaging α-syn aggregates. This work outlines the synthesis of a radiotracer based on the original structure of anle138b, labelled with fluorine-18 isotope, eminently suitable for PET imaging due to half-life and decay energy characteristics (97% β+ decay, 109.7 min half-life, and 635 keV positron energy). A three-step radiosynthesis was developed starting from 6-[18F]fluoropiperonal (6-[18F]FP) that was prepared using (piperonyl)(phenyl)iodonium bromide as a labelling precursor. The obtained 6-[18F]FP was used directly in the condensation reaction with tosylhydrazide followed by 1,3-cycloaddition of the intermediate with 3′-bromophenylacetylene eliminating any midway without any intermediate purifications. This one-pot approach allowed the complete synthesis of [18F]anle138b within 105 min with RCY of 15 ± 3% (n = 3) and Am in the range of 32–78 GBq/µmol. The [18F]fluoride processing and synthesis were performed in a custom-built semi-automated module, but the method can be implemented in all the modern automated platforms. While there is definitely space for further optimization, the procedure developed is well suited for preclinical studies of this novel radiotracer in animal models and/or cell cultures.


Introduction
Positron emission tomography (PET) is a sensitive and versatile imaging modality, often used in conjunction with CT or MRI, offering unique opportunity for a dynamic 3Dvisualization of in vivo processes with a mm-scale resolution. The method employs molecular probes labeled with short-lived positron-emitting radionuclides (PET-radiotracers) interacting with specific protein targets of interest. Apart from widespread diagnostic application in oncology, PET has become an essential tool for assessing pathological processes in a variety of neurodegenerative disorders [1]. Parkinson disease (PD) is the second most widespread neurodegenerative condition worldwide, the prevalence of which is increasing as population ages. PD is characterized by progressive degeneration of the dopaminergic system with loss of dopaminergic neurons in the substantia nigra, associated with clinical presentation of motor symptoms. For in vivo assessment of the central dopaminergic function, the 6-L-[ 18 F]fluoro-3,4-dihydroxyphenyl-alanine has been seen as the "gold standard" since 1983 [2]. Following metabolic pathway of L-DOPA, the The first PET tracer based on the lead structure from the library of the reported 3,5diarylpyrazoles [18] has been the carbon-11 labelled anle253b-a molecule with a suitable position for 11 C-labelling [23]. Evaluation of the radiotracer in healthy rats showed low brain uptake and suboptimal pharmacokinetics [23]. Soon after, the same group introduced a derivative of anle253b called MODAG-001 [24], in which one of the phenyl groups was replaced with pyridine. PET imaging in mice showed excellent brain uptake but detected the formation of two brain-penetrating radio-metabolites-something that hampers quantification of [ 11 C]MODAG-001 uptake. To inhibit the metabolic demethylation process, a deuterated derivative (d3)-[ 11 C]MODAG-001 was developed and was shown to be able to bind to pre-formed α-syn fibrils (α-PFF) in a protein deposition rat model [25]. However, no evidence of binding to aggregated α-syn was observed in human brain sections from DLB patients. Further evaluation of (d3)-[ 11 C]MODAG-001 in a porcine brain with intracerebral injection of α-PFF and post-mortem human AD revealed that the radiotracer was not very selective for α-syn and exhibited significant binding in the AD regions [25].
Despite mixed results of anle138b/MODAG development when it came to carbon-11 labelling [24,25], we considered it worthwhile to attempt synthesis of a radiotracer based on the original structure of anle138b but labelled with longer-lived fluorine-18 isotope (T1/2 110 min) which displays excellent decay characteristics for PET imaging (97% β+ decay, 635 keV positron energy). The structure-activity (SAR) studies revealed that the placement of bromine in meta-position of the 5-phenyl ring led to the highest inhibitory activity of anle138b, whereas further modification of that part of the molecule may result in reduced inhibition [18]. Therefore, in the present work, the 3-substituted aryl moiety was chosen as a suitable labelling position for [ 18 F]anle138b ( Figure 1). To introduce fluorine-18 into this non-activated position of the aromatic ring, a three-step radiolabelling strategy was attempted starting from 6-[ 18 F]fluoropiperonal, obtained through different radiolabelling approaches. As suitability for automation is one of the most important requirements for the labelling method application, a one-pot procedure without intermediate purifications was developed and implemented in a semi-automated module. As a result, [ 18 F]anle138b was obtained in 15.1 ± 2.3% (n = 3) radiochemical yield (decay-corrected) and Am in the range of 31.5-79.5 GBq/µmol within a total synthesis time of ca. 105 min.

Radiolabeling Approach for [ 18 F]Anle138b
The most common method for introduction of fluorine-18 into majority of PET radiotracers is an aliphatic nucleophilic substitution reaction between no-carrier-added [ 18   Despite mixed results of anle138b/MODAG development when it came to carbon-11 labelling [24,25], we considered it worthwhile to attempt synthesis of a radiotracer based on the original structure of anle138b but labelled with longer-lived fluorine-18 isotope (T 1/2 110 min) which displays excellent decay characteristics for PET imaging (97% β+ decay, 635 keV positron energy). The structure-activity (SAR) studies revealed that the placement of bromine in meta-position of the 5-phenyl ring led to the highest inhibitory activity of anle138b, whereas further modification of that part of the molecule may result in reduced inhibition [18]. Therefore, in the present work, the 3-substituted aryl moiety was chosen as a suitable labelling position for [ 18 F]anle138b ( Figure 1). To introduce fluorine-18 into this non-activated position of the aromatic ring, a three-step radiolabelling strategy was attempted starting from 6-[ 18 F]fluoropiperonal, obtained through different radiolabelling approaches. As suitability for automation is one of the most important requirements for the labelling method application, a one-pot procedure without intermediate purifications was developed and implemented in a semi-automated module. As a result, [ 18 F]anle138b was obtained in 15.1 ± 2.3% (n = 3) radiochemical yield (decay-corrected) and A m in the range of 31.5-79.5 GBq/µmol within a total synthesis time of ca. 105 min.

Radiolabeling Approach for [ 18 F]Anle138b
The most common method for introduction of fluorine-18 into majority of PET radiotracers is an aliphatic nucleophilic substitution reaction between no-carrier-added [ 18 F]fluoride and a precursor possessing a suitable leaving group in the presence of phasetransfer catalyst (PTC) to enhance [ 18 F]fluoride reactivity. For aromatic compounds, the classical S N Ar method requires presence of a leaving group as well as an electron-withdrawing group, preferably in orthoor para-position [26]-a requirement difficult to accommodate or engineer in case of complex compounds, such as anle138b. Therefore, the labelling often necessitates multi-steps "built-up" procedures where fluorine-18 is initially introduced into the aromatic ring of a simple and reactive substrate. All nucleophilic fluorinations begin with isolation of [ 18  separately as salts (e.g., HCO 3 , tosylate, triflate); use of such phase-transfer catalysts can simplify trapping/elution procedures [27].
withdrawing group, preferably in ortho-or para-position [26]-a requirement difficult to accommodate or engineer in case of complex compounds, such as anle138b. Therefore, the labelling often necessitates multi-steps "built-up" procedures where fluorine-18 is initially introduced into the aromatic ring of a simple and reactive substrate. All nucleophilic fluorinations begin with isolation of [ 18 F]fluoride ion from proton-irradiated [ 18 O]water; typically, this is achieved by trapping [ 18 F]F − on a quaternary ammonium ion-exchange resin followed by elution with a basic solution of a phase-transfer agent (e.g., Kryptofix K2.2.2/K2CO3 mixture in CH3CN/H2O). The [ 18 F]fluoride ion complex with the PTC can then be dried to remove water and provide a reactive intermediate. In some instances, bulky counter-ions used for solubilisation of the [ 18 F]fluoride, such as R4N + (R = Et or nBu), are introduced separately as salts (e.g., HCO3, tosylate, triflate); use of such phase-transfer catalysts can simplify trapping/elution procedures [27].
Based on this proof-of-concept study, we have focused our efforts on the development of a simplified one-pot labelling procedure for [ 18 (Zarrad, 2017) [29]; RCY-radiochemical yield, decay-corrected.

Synthesis of 6-[ 18 F]FP via Copper-Mediated 18 F-Fluorodeboronation (Method B)
As a starting point, we investigated the feasibility of a copper-mediated approach using the commercially available catalyst Cu(py)4(OTf)2, originally developed for radiofluorination of pinacol arylboronates (ArylBPin) [34]. Its use was further extended to fluorination of organoborons [35] and (hetero)aryl organostannanes [36] and has been found to be useful in the preparation of a wide range of radiotracers [33]. As this methodology has been taken up for the labelling of various precursors, a number of factors have been shown to influence this complex catalytic process. Among them are the Based on this proof-of-concept study, we have focused our efforts on the development of a simplified one-pot labelling procedure for [ 18  Taking into consideration the recent advances in radiofluorination of electron-rich aromatic strictures [30][31][32][33], we explored alternative routes to 6-[ 18 F]FP using different radiolabelling precursors and conditions ( Figure 2B-D).

Synthesis of 6-[ 18 F]FP via Copper-Mediated 18 F-Fluorodeboronation (Method B)
As a starting point, we investigated the feasibility of a copper-mediated approach using the commercially available catalyst Cu(py) 4 (OTf) 2 , originally developed for radiofluorination of pinacol arylboronates (ArylBPin) [34]. Its use was further extended to fluorination of organoborons [35] and (hetero)aryl organostannanes [36] and has been found to be useful in the preparation of a wide range of radiotracers [33]. As this methodology has been taken up for the labelling of various precursors, a number of factors have been shown to influence this complex catalytic process. Among them are the sensitivity of the Cu-mediated process to the basic conditions during PTC-controlled solubilisation of [ 18 F]fluoride, reaction solvents used, precursor/copper catalyst ratio and others [30,[37][38][39].
Using the two labelling precursors-2a and 2b-we applied our previously developed [ 18 F]fluoride trapping-elution protocol by replacing Et 4 NHCO 3 [29] with the non-basic Bu 4 NOTf (10 µmol) in conjunction with 2-PrOH (0.6 mL) as the eluting solvent [41,42]. The fluoride-PTC complex was eluted directly into the reaction vial containing the labelling precursor and Cu(py) 4 (OTf) 2 in a suitable solvent (0.8 mL), avoiding any evaporation steps. Optimisation of radiofluorination parameters in terms of the solvents used and amounts of the reactants was realized using commercially available boronic acid derivative 2a ( Figure 2). From several solvents investigated (Table 1), the highest fluorination (RCC of 96 ± 2%, Table 1, Entry 5) was achieved carrying out radiofluorination in the mixture of 2-PrOH/CH 3 CN (2/3) at precursor-to-copper catalyst ratio of 20/20 µmol. This protocol worked equally for radiofluorination of ArylBPin precursor 2b, providing the desired 6-[ 18 F]FP with RCC of >97%. For practical reasons, the use of 2a as a commercially available precursor would of course be preferable. Further reduction in the reactants amounts down to 10/10 µmol has resulted in ca. 50% decrease in the yield of radiofluorination of 2a (Table 1, Entry 6). No product formation could be observed when radiofluorination of 2a was carried out in DMA, a solvent typically used in Cu-mediated radiofluorinations, with 2-PrOH as co-solvent (Table 1, Entry 1). The relatively low conversion rates of 2a (Table 1, Entries 3, 4) were accompanied by substantial losses of radioactivity on the inner surfaces of the reaction vessel (up to 70% of total radioactivity in the case of neat 2-PrOH) and substantial RCC variability. This could be explained by poor solubility of the labelling precursor in the solvents used.

Synthesis of 6-[ 18 F]FP via Diaryliodonium Salts Precursors (Methods C and D)
Another effective approach to the incorporation of [ 18 F]fluoride into (hetero)aromatic substrates is the use diaryliodonium (DAI) salt precursors-route pioneered by the Pike group [43]. A very practical approach for performing radiofluorination has been introduced for the onium and DAI salts precursors [44]. The advantageous feature of this approach is that the [ 18 F]fluoride retained on the anion-exchange resin is eluted directly with the solution of the DAI precursor in MeOH; following quick removal, the solvent is then directly followed by a fluorination reaction. This type of procedure uses neither the PTC/base nor other additives, it reduces the number of operational steps, saves time and is compatible with base-sensitive precursors and products.
A copper-mediated radiofluorination of (mesityl)(aryl)iodonium (MAI) salts using the commercially available (CH 3 CN) 4 CuOTf complex and [ 18 F]KF/18-crown-6 for the activation of the fluorine-18 was suggested in 2014 [45] as an effective route for radiofluorination of the electron rich arenes. It was shown that when involving the use of a bulky mesityl group as an auxiliary force, the nucleophilic substitution acted towards the less sterically hindered site on the arene ring [45]. A recently developed protocol has been applied for the synthesis radiotracers using MAI salts as labelling precursors [46]. In brief, [ 18 F]fluoride is eluted from the anion-exchange matrix with solution of MAI precursor in MeOH/DMF (20% MeOH), followed by Cu-mediated fluorination in the same solvent mixture. Despite the high [ 18 F]F − elution and fluorination efficiency achieved for a series of 18 F-fluorinated aromatic amino acids [46], utilising this approach for the preparation of 6-[ 18 F]FP from MAI salt 3 ( Figure 2) resulted in elution efficiency from anion-exchange matrix using 3 (20 µmol) in 20% MeOH/DMF (0.72 mL) being low, as was the radiofluorination reaction yield ( (1) 75-85/10 (2) 120/20 (1) 75-85/10 (2) 120/20 Consequently, we moved on to the more conventional approach for nucleophilic radiofluorination using tetraalkylammonium salts as PTCs for the activation of [ 18 F]fluoride. Radionuclide was eluted from the cartridge using 4 µmol of Et 4 NHCO 3 or Bu 4 NOTs in 1 mL of MeOH followed by the solvent evaporation. The reactive [ 18 F]fluoride thus obtained was allowed to react with 3 in the presence of the Cu(I)-catalyst in DMF, affording 6-[ 18 F]FP in the RCC of ca. 40-50% when high amounts of the reactants were used ( Table 2, Entries 4 and 6). However, despite the improvement in radiofluorination efficiency, the radioactivity yield was not high enough considering the desired 6-[ 18 F]FP is an intermediate in a multistep synthesis.
A second strategy which does not involve transition metal catalysts is the radiofluorination of the (phenyl)iodonium salts precursors 4a and 4b with different counter ions ( Figure 2). Starting with the tosylate salt 4a, we failed to produce 6-[ 18 F]FP consistently using either approach described earlier (  Figure S18) in radiofluorination of bromide iodonium salt 4b (Table 2, Entry 16) when performing radiofluorination with two-step heating sequence. Introduction of such a heating procedure was based on our UV-HPLC analysis of samples of the reaction mixture that revealed decomposition of 4b during the MeOH evaporation step ( Table 2, Entry 15). To prevent precursor decomposition, MeOH was removed during the first heating step at 75-85 • C under gentle agitation with nitrogen gas flow, while the radiofluorination step was performed at 120 • C for 20 min in a neat propylene carbonate (PC), affording 6-[ 18 F]FP in high RCC. However, using the same protocol, only around 3% RCC was observed in the radiofluorination of the tosylate salt 4a (  [47]. We assume that the radiofluorination might be suppressed by the presence of residual silver in 4a that was prepared from 4b by reaction with silver tosylate. Finally, from all of the investigated radiolabeling procedures, highest RCCs for 6-[ 18 F]FP were achieved with Cu-mediated radiofluorination of commercially available 2a using [ 18 F]Bu 4 NOF in 2-PrOH/CH 3 CN (Table 1, Entry 5). However, the radiofluorination of (phenyl)iodonium salt 4b in MeOH/PC (Table 2, Entry 15) avoiding Cu-catalyst turned out to be most suitable route for preparing 6-[ 18 F]FP with additional benefits for the synthesis automation through the use of a fairly simple and straightforward trapping/elution protocol for [ 18 F]F − (elution efficiency was over 85%).

Radiosynthesis of [ 18 F]Anle138b
Radiolabeling of [ 18 F]anle138b starting from 4b as a labelling precursor is depicted in Scheme 2.
that revealed decomposition of 4b during the MeOH evaporation step (Table 2, Entry 15). To prevent precursor decomposition, MeOH was removed during the first heating step at 75-85 °C under gentle agitation with nitrogen gas flow, while the radiofluorination step was performed at 120 °C for 20 min in a neat propylene carbonate (PC), affording 6-[ 18 F]FP in high RCC. However, using the same protocol, only around 3% RCC was observed in the radiofluorination of the tosylate salt 4a (Table 2, Entry 14). Such a low radiofluorination efficiency of 4a, as compared to the bromide salt 4b, is not supported by the literature data demonstrating that tosylate is one of the most reactive salts towards the [ 18 F]fluoride ion [47]. We assume that the radiofluorination might be suppressed by the presence of residual silver in 4a that was prepared from 4b by reaction with silver tosylate.
Finally, from all of the investigated radiolabeling procedures, highest RCCs for 6-[ 18 F]FP were achieved with Cu-mediated radiofluorination of commercially available 2a using [ 18 F]Bu4NOF in 2-PrOH/CH3CN (Table 1, Entry 5). However, the radiofluorination of (phenyl)iodonium salt 4b in MeOH/PC (Table 2, Entry 15) avoiding Cu-catalyst turned out to be most suitable route for preparing 6-[ 18 F]FP with additional benefits for the synthesis automation through the use of a fairly simple and straightforward trapping/elution protocol for [ 18 F]F − (elution efficiency was over 85%).

Radiosynthesis of [ 18 F]Anle138b
Radiolabeling of [ 18 F]anle138b starting from 4b as a labelling precursor is depicted in Scheme 2. When developing our method for [ 18 F]anle138b, we were, in a significant part, guided by the previously outlined synthetic strategy [29], while focusing our attention on making the synthesis as simple as possible by eliminating intermediate purification steps and employing the one-pot approach. The [ 18 F]fluoride isolation and synthesis itself were conducted in a custom built synthesis module, described in the experimental part. For monitoring of radiolabelling progress, aliquots of the relevant reaction mixture were taken after each synthesis step (Scheme 2) and analyzed by radio-HPLC (Figure 3). When developing our method for [ 18 F]anle138b, we were, in a significant part, guided by the previously outlined synthetic strategy [29], while focusing our attention on making the synthesis as simple as possible by eliminating intermediate purification steps and employing the one-pot approach. The [ 18 F]fluoride isolation and synthesis itself were conducted in a custom built synthesis module, described in the experimental part. For monitoring of radiolabelling progress, aliquots of the relevant reaction mixture were taken after each synthesis step (Scheme 2) and analyzed by radio-HPLC ( Figure 3).
As described above, the first synthesis step-the radiofluorination of aryliodonium bromide 4b afforded 6-[ 18  Remarkably, the efficiency of the cycloaddition reaction greatly depends on the nature of base employed in the previous step. From several bases investigated (DBU, NaOH, K 2 CO 3 and LiOtBu) [29], the highest conversion rate of ca. 50% was achieved using LiOtBu. Unfortunately, LiOtBu is insoluble in most organic solvents, including PC that was an essential component of the reaction mixture in our one-pot three-step synthesis procedure. To address this issue, Bu 4 NOH soluble in both CH 3 CN and PC was used as a base. Furthermore, according to the data previously reported [48], the efficiency of one-pot cycloaddition process could be improved by sequential introduction of the base and corresponding acetylene into reaction mixture. Taking those considerations into account, we adjusted our procedure to employ sequential addition of reagents in the following order: The solution of Bu 4 OH in CH 3 CN was added first with heating at 65 • C for 5 min, followed by 3 -bromophenylacetylene addition and a second round of heating at 90 • C for 25 min. With this approach, we were able to obtain [ 18 F]anle138b in the RCC of 60% ( Figure 3C [29], the highest conversion rate of ca. 50% was achieved using LiOtBu. Unfortunately, LiOtBu is insoluble in most organic solvents, including PC that was an essential component of the reaction mixture in our one-pot three-step synthesis procedure. To address this issue, Bu4NOH soluble in both CH3CN and PC was used as a base. Furthermore, according to the data previously reported [48], the efficiency of one-pot cycloaddition process could be improved by sequential introduction of the base and corresponding acetylene into reaction mixture. Taking those considerations into account, we adjusted our procedure to employ sequential addition of reagents in the following order: The solution of Bu4OH in CH3CN was added first with heating at 65 °C for 5 min, followed by 3′-bromophenylacetylene addition and a second round of heating at 90 °C for 25 min. With this approach, we were able to obtain [ 18 F]anle138b in the RCC of 60% ( Figure  3C) using 0.4 mmol 3′-bromophenylacetylene and 26 µmol Bu4NOH. For the identification of the target product, the authentic reference standard [ 19 F]anle138b was prepared (Scheme 3); the synthesis is described in the experimental part.

Purification and Quality Control of [ 18 F]Anle138b
Due to high lipophilicity of anle138b, isolation of the 18 F-fluorinated derivative by conventional semi-preparative HPLC presents a challenge. Given the "one-pot" synthesis approach and the resulting multicomponent reaction mixture, the purification task becomes even more complicated.

Purification and Quality Control of [ 18 F]Anle138b
Due to high lipophilicity of anle138b, isolation of the 18 F-fluorinated derivative by conventional semi-preparative HPLC presents a challenge. Given the "one-pot" synthesis approach and the resulting multicomponent reaction mixture, the purification task becomes even more complicated.
The isolation of [ 18 F]anle138b from the reaction mixture for two different HPLC systems was evaluated. The first one (System A) is an integral part of the GE Tracerlab FX C Pro module, equipped with UV-and β-radioactivity flow detectors. The HPLC separation was performed on a reverse-phase Ascentis RP-Amide semi-preparative column, 250 × 10 mm (Sigma-Aldrich GmbH, Steinheim, Germany). The content of the reaction vessel (1.2 mL) was diluted with the mobile phase and transferred to the 2 mL injection loop. For practical reasons, biocompatible ethanol-containing eluent would be preferable. However, when using H 2 O/EtOH gradient system (gradient conditions 1, Materials and Methods) at a flow rate of 3.0 mL/min, only 5% of the injected radioactivity was recovered as [ 18 F]anle138b (R t of 25-26 min) with radiochemical purity (RCP) in order of 80%. Replacing EtOH with CH 3 CN in the mobile phase and modifying gradient (gradient conditions 2, Materials and Methods) afforded [ 18 F]anle138b in 90% RCP with slightly increased recovery of the radioactivity (8%) in the product fraction (R t of 28-32 min).
Alternatively, we investigated applicability of a different HPLC column, Chromolith SemiPrep RP-18e, 100 × 10 mm equipped with a UV and radioactivity detector, a gradient pump and the Rheodyne-type injector with a 100 µL loop (System B). Under gradient conditions using EtOH-based mobile phase at a flow rate of 4 mL/min, the [ 18 F]anle138b (fraction with R t 9.7-9.9 min, 1 mL volume, 26% recovery of the product) was obtained in more than 98% RCP according to radioHPLC (Figure 4), and ca. 100% according to radioTLC (Supplementary Materials, Figure S19) As the ethanol-containing eluent was applied, cartridge based reformulation of the product was not required.  Recalculating the radioactivity of the volume injected (100 µL) to total reaction volume (1.2 mL), decay-corrected RCY of [ 18 F]anle138b was 15.1 ± 2.3% (n = 3) with synthesis time of ca. 105 min. Am was in the range of 31.5-79.5 GBq/µmol.

General Chemistry
All commercially available chemicals were used without any further purification. The main limitation of this method is a small maximum injection volume (100 µL); with higher injected volumes, the column performance would degrade due to overloading. Therefore, multiple injections would be required for purification of all reaction volume produced in the synthesis (1.2 mL total). Nonetheless, the procedure developed is well suited for preclinical studies where small amounts of the radiotracer are typically injected.
Radio-TLC analyses were carried out on silica gel plates (60 F254, Merck or Sorbfil, Lenchrom, Russia); radioactivity distribution was determined using a Scan-RAM radioTLC scanner controlled by the chromatography software package Laura (v6.0.4.92) for PET (LabLogic, Sheffield, UK). An aliquot (2-3 µL) of the crude reaction mixture diluted with acetonitrile, was applied onto a TLC plate, and the plate was then developed in ethyl acetate. The R f values for [ 18 F]fluoride, 6-[ 18 F]FP and [ 18 F]anle138b were 0.05, 0.57 and 0.67, correspondingly. The radiochemical conversion (RCC) measured by radioTLC was defined as the ratio of the product peak area to the total peak area on the TLC. RCC values were not corrected for radioactive decay.
Analytical HPLC was performed on a Dionex ISC-5000 system (Dionex, Sunnyvale, CA, USA). It was equipped with a gradient pump, Rheodyne type injector with a 20 µL loop and a UV absorbance detector with variable wavelength (set to 254 nm) connected in series with a radiodetector (Carrol and Ramsey Associates, CA, USA, model 105-S) giving a delay of 0.1 min. The identity, radiochemical and chemical purity of the [ 18 F]anle138b and analysis of the reaction mixture at each stage of the synthesis were determined under the following HPLC conditions: X-Bridge C18 HPLC column, 150 × 4.6 mm (Waters Corporation, Millford, CT, USA), eluent with 5-95% gradient (0.1% aq. TFA/MeCN), and a flow rate of 2.0 mL/min. Overall, there was a 0-8.0 min 5-95% MeCN linear increase; 8.0-11.0 min 95% MeCN isocratic; 11.0-11.2 min 95-5% MeCN linear decrease; and an 11.2-15.0 min 5% MeCN isocratic. The R t values for the precursor, reference and radiolabelled intermediates are presented in Table 3.  (Figure 2). Cu-mediated radiofluorination of pinacol arylboronates (2a, 2b). A solution of [ 18 F]F -(0.5-1.0 GBq) in [ 18 O]H 2 O was loaded from the male side onto a QMA cartridge. The cartridge was flushed from the male side with 2-PrOH (4 mL) and dried with N 2 gas for 2 min. 18 Fwas eluted from the female side of the cartridge with a solution of Bu 4 NOTf (4 mg, 10 µmol) in 2-PrOH (0.6 mL) into the 2 mL reaction vessel prefilled with a solution of 2a or 2b (20 µmol) and Cu(py) 4 (OTf) 2 (20 µmol) in MeCN (0.8 mL). The reaction mixture was heated at 65 • C for 10 min, followed by a second round of heating 110 • C for 10 min, while the reactor was sealed via valve 16 ( Figure 5). Then, the reaction vessel was cooled down to 40 • C.
A solution of [ 18 F]F -(0.5-1.0 GBq) in [ 18 O]H 2 O was loaded from the male side onto a QMA cartridge. The cartridge was flushed from the male side with MeOH (2 mL) and dried with N 2 gas for 2 min. 18 Fwas eluted from the female side of the cartridge with a solution of Et 4 NHCO 3 (0.8 mg, 4.2 µmol) in MeOH (1 mL) into the 2 mL reaction vessel. The solvent was evaporated by heating to 75 • C under gas flow, and the reaction vessel was then cooled to 50 • C. The solution of 3 (30 µmol) and Cu(MeCN) 4 OTf (30 µmol) in DMF (0.5 mL) was then added to the dried residue and the reaction mixture, and the reaction mixture was heated at 90 • C for 20 min, while the reactor was sealed via valve 16 ( Figure 5). Then, the reaction vessel cooled down to 40 • C. dried with N2 gas for 2 min. 18 Fwas eluted from the female side of the cartridge with a solution of Bu4NOTf (4 mg, 10 µmol) in 2-PrOH (0.6 mL) into the 2 mL reaction vessel prefilled with a solution of 2a or 2b (20 µmol) and Cu(py)4(OTf)2 (20 µmol) in MeCN (0.8 mL). The reaction mixture was heated at 65 °C for 10 min, followed by a second round of heating 110 °C for 10 min, while the reactor was sealed via valve 16 ( Figure 5). Then, the reaction vessel was cooled down to 40 °C.  was loaded from the male side on a QMA cartridge. The cartridge was flushed from the male side with MeOH (2 mL) and dried with N 2 gas for 2 min. 18 Fwas eluted from the female side of the cartridge with a solution of the respective radiolabelling precursor 4a or 4b (20 µmol) in 44% MeOH/PC (0.9 mL). The reaction mixture was heated at 85 • C for 10 min with stirring N 2 followed by the second round of heating at 120 • C for 20 min, while the reactor was sealed via valve 16 ( Figure 5). The reaction vessel was cooled down to 40 • C.

Synthesis of [ 18 F]Anle138b from 4b (Scheme 2)
Step 1. Radiofluorination of diaryliodonium salt 4b (Method D). A solution of [ 18 F]F -(9.0-11.0 GBq) in [ 18 O]H 2 O was loaded onto a QMA cartridge from the male side followed by flushing with MeOH (2 mL) and drying with N 2 gas for 2 min. 18 Fwas eluted from the female side of the cartridge with a solution of 4b (20 µmol) in 44% MeOH/PC (0.9 mL). The reaction mixture was heated at 85 • C for 10 min with stirring by nitrogen flow followed by the second round of heating at 120 • C for 20 min, while the reactor was sealed via valve 16 ( Figure 5). The reaction vessel was cooled down to 40 • C.
The solution of NH 2 NHTs (40 µmol) in MeOH (1 mL) was added to the reaction mixture obtained at step 1; the content was heated for 10 min at 90 • C under stirring by nitrogen flow. The reaction vessel was cooled down to 65 • C.
Step 3. Synthesis of [ 18 F]anle138b. The solution of Bu 4 NOH (26 µmol) in MeCN (0.4 mL) was added to the reaction mixture obtained at step 2 and the content was heated for 5 min at 65 • C. Then the solution of 3 -bromophenylacetylene (0.4 mmol) in MeCN (0.4 mL) was added and the reaction mixture was heated for 25 min at 90 • C. The reaction vessel was cooled down to 40 • C.

HPLC Purification
System A. The content of the reaction vessel (1.2 mL volume) was diluted with 0.8 mL 50% EtOH or with 0.8 mL of CH 3 CN. The resulting solution was transferred to 2 mL HPLC loop. The fraction containing [ 18 F]anle138b (R t of 25-26 min under gradient 1 conditions or 28-32 min under gradient 2 conditions) was collected and analysed for radiochemical and chemical purity.
System B. The aliquot (100 µL) of the reaction mixture (from 1.2 mL total volume) was loaded onto 100 µL of HPLC loop. The fraction containing [ 18 F]anle138b (R t of 9.7-9.9 min, 1 mL volume) was collected through a 0.22 µm filter (Millipore, Burlington, MA, USA) attached to a vented sterile vial prefilled with the formulation buffer.

Semi-Automated Synthesis of [ 18 F]Anle138b (Figure 5)
The [ 18 F]fluoride processing and all the synthesis steps were completed in a custombuilt synthesis module described in detail elsewhere [46]. The reactions were performed in a 5 mL V-vial (RV, Figure 5) with a screw cap (Wheaton-vials, Sigma-Aldrich GmbH, Steinheim, Germany). Nitrogen gas was applied for the reagents transfer.
in preclinical studies in animal or cell models. In addition, the suggested methodology may find further use in the preparation of other PET imaging agents derived from the pyrazoles backbone.